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Dynamin recruitment by clathrin coats: a physical step? — Recrutement des dynamines par les puits recouverts de clathrines : une étape physique? J.-B. Fournier∗ and P.-G. Dommersnes† Laboratoire de Physico-Chimie Théorique and FR CNRS 2438 “Matière et Systèmes Complexes”, ESPCI, 10 rue Vauquelin, F-75231 Paris cedex 05, France 3 P. Galatola 0 LBHP, Université Paris 7—Denis Diderot and FR CNRS 2438 “Matière et Systèmes Complexes”, 0 Case 7056, 2 place Jussieu, F-75251 Paris cedex 05, France 2 (Dated: February 2, 2008) n a Abstract – Recent structural findings have shown that dynamin, a cytosol protein playing a J key-role in clathrin-mediated endocytosis, inserts partly within the lipid bilayer and tends to self- 5 assemblearoundlipidtubules. Takingintoaccounttheseobservations,wemakethehypothesisthat 1 individualmembraneinserteddynaminsimprintalocalcylindricalcurvaturetothemembrane. This imprint may give rise to long-range mechanical forces mediated by the elasticity of the membrane. ] t Calculating the resulting many-body interaction between a collection of inserted dynamins and a f o membranebud,wefindaregimeinwhichthedynaminsareelasticallyrecruitedbythebudtoform s acollararounditsneck,whichisreminiscentoftheactualprocesspreemptingvesiclescission. This . physical mechanism might therefore beimplied in therecruitment of dynaminsby clathrin coats. t a m endocytosis / clathrin / dynamin / membrane inclusions interactions - d Résumé–Desdonnésstructuralesrécentesontmontréqueladynamine,uneprotéineducytosol n quijoue unrôle clé dansl’endocytose clathrine-dépendante,s’insère partiellement dansla bicouche o membranaire et tend à s’auto-assembler autour de tubules lipidiques. En tenant compte de ces c observations, nousfaisons l’hypothèsequelesdynaminesimprimentlocalement unecourburecylin- [ drique dans la membrane. Cette empreinte peut engendrer des forces élastiques de longue portée. En calculant l’interaction multi-corps entre un ensemble de dynamines insérées dans la membrane 1 et une capsule endocytotique, nous trouvons un régime dans lequel les dynamines sont recrutées v élastiquement par la capsule pour former un collier autour de son cou, ce qui rappelle le processus 7 précédantlascissiondesvésiculesd’endocytose. Cemécanismephysiquepourraitdoncêtreimpliqué 5 dans le recrutement des dynaminespar les capsules declathrine. 2 1 endocytose / clathrine / dynamine / interactions entre inclusions membranaires 0 3 0 / t a I. INTRODUCTION m - Ineukaryoticcells,membranesofdifferentorganellesarefunctionallyconnectedtoeachotherviavesicular d n transport. Formationoftransportvesiclesfrominvaginatedbudsoftheplasmamembraneiscalledendocyto- o sis [1]. Inclathrin-mediatedvesiculation,vesicleformationstartswiththeassemblyonthedonormembrane c ofahighlyorganized“coat”ofclathrins[2],whichactsbothtoshapethemembraneintoabudandtoselect : v cargo proteins [3, 4, 5, 6, 7]. The mechanism by which an invaginated clathrin-coatedbud is convertedto a i vesicle(scission)involvesthe actionof a cytoplasmicGTPaseproteincalleddynamin [6, 8]. Dynamins form X oligomeric rings at the neck of deeply invaginated membrane buds and induce scission [9, 10]. How exactly r dynaminis recruitedandhowthescissionactuallyoccursremainsunclear[11, 12]. Inthis paperwepropose a that dynamin recruitment by clathrin coats could be driven by long-rangedphysical forces mediated by the membrane curvature elasticity. Cryo-electronmicroscopyhas recently revealedthe detailed structure of the clathrin coats at 21˚Aresolu- tion[2]. Clathrinunits,alsocalled“triskelions”,haveastar-likestructurewiththreelegs. Initiallysolubilized ∗Authorforcorrespondence([email protected]) †Presentaddress: InstitutCurie,UMR168,26rued’Ulm,F-75248ParisCédex05,France. 2 PROTEIN PSfragrepla ements binding inserting MEMBRANE FIG. 1: Schematic representation of a cytosol protein partly inserting within a lipid bilayer and inducing a local membrane curvaturevia a binding region. intothecytoplasmicfluid,theyself-assembleontothemembranesurfaceintoacurved,two-dimensionalsolid scaffold. Thelatterisahoneycombmadeofhexagonsandpentagons(geometricallyprovidingthecurvature) the sides of which are built by the overlapping legs of the clathrin triskelions. In the plasma membrane, clathrins usually interactwith “adaptor”transmembraneproteins, whichalso serveto select cargoproteins. Howeverit has been shownthat clathrin coatscanreadily self-assembleonto protein-freeliposomes [13, 14]. Dynamin is known to be solubilized in the cytosol as tetramers, and to aggregate in low-salt buffers into rings and spirals [9]. Dynamin also self-assembles onto lipid bilayers,forming helically striated tubules that resemblethenecksofinvaginatedbuds(tubediameter≃50nm)[10]. AdditionofGTPinducesmorphological changes: either the tubules constrict and break [15], or the dynamin spiral elongates [16]. These findings suggest that the scission of clathrin-coated buds is produced by a mechanochemical action [16, 17]. At the earlier stages of the budding process, dynamins already strongly interact with bilayer membranes. Indeed, in vivo studies showed that dynamin binds acidic phospholipids in a way that is essential to its ability to form oligomeric rings on invaginated buds [18, 19, 20, 21, 22]. Using a model lipid monolayer spread at the air-water interface, it was shown that dynamins actually penetrate within the acyl region of the membrane lipids [23]. This finding was recently confirmed by the three-dimensional reconstruction of the dynamin structure by cryo-electronmicroscopyat20˚Aresolution[24]: dynamins formT-shapeddimers the “leg” of which inserts partly into the outer lipid leaflet. Itwaslongagosuggested[25,26]thatparticlesinsertedwithinbilayersshouldfeellong-rangeinteractions mediated by the elasticity of the membrane. Indeed, a protein penetrating within a bilayer and binding its lipids—such as dynamin—may in general produce a local membrane curvature (see Fig. 1). Because of the very nature of the curvature elasticity of fluid membranes, this deformation relaxes quite slowly away from its source, and the presence of another inserted particle produces an interference implying an interaction energy [25]. This holds as long as the separation between the inclusions is smaller than the characteristic length ξ = κ/σ, where κ ≃ 60k T is the bending rigidity of the membrane and σ is the tension of σ B the membrane. At separations larger than ξ , the membranes flattens out and the interaction vanishes σ p exponentially. Note that the membrane tension is not a material constant like κ; it is an effective force per unitarea,whichismostprobablybiologicallyregulated[27]andisusuallyoftheorderof10−2 to10−5 times the surface tension of ordinary liquids [27, 28]. At this point, anticipating on our model for the clathrin- dynaminsystem, let us state that we shallformallyassume σ =0 inthis paper, whichamountsto assuming that the relevant distances between the inclusions (i.e., the distance between the neck of the clathrin bud and the dynamins) are smaller than ξ . This means that our model should rather apply to weakly tense σ membranes, e.g., σ ≃ 10−3mJ/m2, for which ξ ≃ 400nm (which is quite larger than the typical size of σ the clathrin buds ≃ 80nm). Although the actual value of σ in the vicinity of clathrin buds is unknown, such a small tension agrees with recent measurements on biological membranes (erythrocytes membranes interacting with their cytoskeleton) [28]. In the case of stronger tensions, we expect our results to hold nonetheless, the dynamins being “captured” when their Brownian diffusion brings them at a distance from the bud less than ξ . σ The first detailed calculation of the membrane mediated interaction was performed for two isotropic particles each locally inducing a spherical curvature [29, 30]. The interaction was found to be repulsive, proportional to the rigidity κ of the membrane and to the sum of the squares of the imposed curvatures; it decays as R−4, where R is the distance between the particles. The case of anisotropic particles producing non-sphericalmembranedeformationsisevenmoreinteresting,sincetheircollectiveactiononthemembrane isexpectedtohavenontrivialmorphologicalconsequences[31,32,33]. Thelocaldeformationofamembrane actually involves two distinct curvatures, associated with two orthogonal directions (as in a saddle or in a cylinder). Recent calculations showed that the interaction between two anisotropic inclusions is very long- rangedand decaysas R−2 [34, 35, 36]. It is alwaysattractiveat largeseparationsand favorsthe orientation of the axis of minor curvature along the line joining the particles [35]. Note that these elastic interactions prevail atlarge separations,since they are of much longer rangethan other forces,such as van der Waals or 3 FIG. 2: Piece of a model membraneshowing a clathrin coated bud and theimprints of inserted dynamins. screened electrostatic interactions. II. MODEL Amongtheaboveinformations,letusoutlinethethreepointsthatareessentialforourmodel. (i)Clathrin coatsaresolidscaffoldsthatrigidlyshapeextendedpartsofthemembraneintosphericalcaps. (ii)Dynamins are solubilized proteins that partly insert within the membrane bilayer. (iii) Inserted membrane hosts that imprint a local membrane curvature interact with long-range forces of elastic origin. We therefore build the following model. We consider a clathrin coated bud as being a membrane patch bearing a constant, fixed spherical curvature. Technically, we shall build the bud by placing a large number of point-like sphericalcurvature sourcesat the vertexof a hexagonallattice (see Fig. 2). Within the present formalism, this is the simplest way to define a rigid, almost non-deformable, spherically curved zone. Since the dynamins will not penetrate the bud, we expect that our results will not depend on whether the bud is geometrically enforced (which would be conceptually simpler but technically harder here) or built by an inclusion array. Note that the shape of the neck around the clathrin bud will result from the minimization of the total energy, and will therefore not be enforced artificially. Because dynamins partly insert within the membrane and seem to accommodate cylindrical curvature, we model them as sources locally imprinting a cylindrical curvature. We place a large number of such “dynamins” on a membrane in the presence of an artificial bud as described above (Fig. 2), and we study whether the latter will recruit or not the dynamins through elastic long-range forces. A. Long-range elastic interactions between many membrane inclusions The elastic interaction between N isotropic or anisotropic membrane hosts can be calculated from first principles [35, 37, 38, 39]. The membrane is described as a surface which is weakly deformed with respect to a reference plane. Without this assumption, analytical calculations are virtually impossible. An obvious consequence is that we can accurately describe only weakly invaginated buds; nevertheless, we expect that our results will hold qualitatively for strongly invaginated buds. The membrane hosts are described as point-like sourcesbearing twocurvaturesc andc , associatedwithtwo orthogonaldirections. These values 1 2 represent the two principal curvatures that the hosts imprint on the membrane through their binding with the membrane lipids (assuming the binding region is itself curved). For instance, a spherical impression corresponds to c /c = 1, a cylindrical impression corresponds to c /c = 0, and a saddle-like impression 1 2 1 2 4 correspondstoc /c =−1. Thisover-simplifiedmodelactuallycontainstheessentialingredientsresponsible 1 2 for the long-range elastic interactions between membrane inclusions: for protein hosts of a size comparable to the thickness of the membrane, it yields accurate interactions for separations as small as about three times the particles size. Note that the curvature actually impressed by a particle could be affected by the vicinity of another inclusion, we shall however neglect this effect for the sake of simplicity (strong binding hypothesis). The point-like curvature sources describing the membrane hosts diffuse and rotate within the fluid membrane, because of the forces and torques exerted by the other membrane hosts and of the thermal agitation k T. We parameterize the orientation of a particle by the angle θ that its axis of minor curvature, i.e., the B axis associatedwith min(|c |,|c |), makes with the x-axis, in projection on the (x,y) reference plane. Given 1 2 N inclusions with specified positions x and y , orientations θ , and curvatures c and c , for i = 1...N, i i i 1i 2i we calculate the shape of the membrane satisfying the N imposed point-like curvatures and we deter- mine the total elastic energy of the system. We thereby deduce the N-body interaction between the hosts F (...,x ,y ,θ ,c ,c ,...). The mathematical details of this procedure are sketched in Appendix A. int i i i 1i 2i B. Pairwise interactions Before studying the collective interaction between model dynamins and clathrin coats, let us describe how point-like spherical and cylindrical sources interact pairwise (in the absence of membrane tension, as discussed in Sec. I). The membrane distortion produced by two inclusions modeled as point-like sphericalcurvature sources is shown in Fig. 3a. Each inclusion appears as a small spherical cap away from which the membrane relaxes to a flat shape. As evidenced by the plot of the interaction energy (see Fig. 3a), such spherical inclusions repel one another. Calling κ the bending rigidity of the membrane,a the thickness of the membrane (which is comparableto the size ofthe inclusions), c the curvature setby the inclusions andR their separation,our calculation gives (see Appendix A): a 4 F (R)≃8πκa2c2 (1) cl−cl R (cid:16) (cid:17) for the the asymptotic interaction at large separations,in agreement with previous works [29, 34, 35]. Note that the plot given in Fig. 3a corresponds to the exact interaction within our model, not to the asymptotic expression (1). The membrane distortion produced by two inclusions modeled as point-like cylindrical curvature sources isshowninFig.3b. Eachinclusionappearsasasmallcylindricalcapawayfromwhichthemembranerelaxes toaflatshape. Theinteractionbetweentwosuchhostsdepends ontheirrelativeorientation. Theminimum energy is found when the axes of the cylinders are parallel to the line joining the inclusions. As evidenced by the plot of the interaction energy (see Fig. 3b), the interaction is then attractive. It therefore turns out thattwosuchhostsproduce aweakermembranedeformationwhentheyarecloseto oneanotherthanwhen they are far apart. As described in Ref. [35], when their curvature is strong enough, such inclusions tend to aggregate and to form linear oligomers. Their asymptotic interaction energy is given by a 2 F (R)≃−8πκa2c2 . (2) dy−dy R (cid:16) (cid:17) It decays as R−2, hence it is of longer range than (1). Finally, we show in Fig. 3c the membrane distortion produced by the interaction between a spherical sourceandacylindricalone. The latter is orientedin the directionthatminimizes the energy. As evidenced by the plot of Fig. 3c, the interaction is attractive at large separations and repulsive at short separations, with a stable minimum configuration at a finite distance. Calling c the curvature set by the cylindrical inclusion and c′ the one set by the spherical inclusion, our calculations give the asymptotic interaction a 2 F (R)≃−4πκa2cc′ . (3) cl−dy R (cid:16) (cid:17) We may therefore expect that dynamins will be attracted by clathrins coats; however, owing to the non- pairwise character of the interaction [35], it is necessary to actually perform the corresponding many-body calculation. It is also necessary to check whether thermal agitation will or will not disorder the inclusions. 5 FIG. 3: Shape of a membrane distorted by two inclusions imprinting local curvatures and interaction energy as a function of separation. Distances are rescaled by the membrane thickness a and energies by κa2c2. (a) spherical inclusionsofcurvaturec;(b)cylindricalinclusionsofcurvaturec;(c)sphericalinclusionofcurvaturecandcylindrical inclusion of curvature0.2c. The shapes are calculated from Eq.(A13), theinteraction energies from Eq. (A12). III. COLLECTIVE INTERACTIONS BETWEEN MODEL DYNAMINS AND CLATHRIN BUDS As described in Sec. II, we build a model clathrin-coated bud by placing in a membrane N point-like cl spherical inclusions of curvature c on a hexagonal array with lattice constant b. Here, we have chosen cl N = 37 and b = 3a. By changing the curvature c , we can adjust the overall curvature of the clathrin cl cl scaffold, thereby simulating the growth of a vesicular bud. We then add N = 40 point-like cylindrical dy sources of curvature c modeling inserted dynamins. dy To study the collective behavior of this system under the action of the multibody elastic interactions (see 6 0.2 b (cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1) R G PSfrag r(cid:0)(cid:1)e(cid:0)(cid:1)p(cid:0)(cid:1)la ements cl (cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1) (cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1) (cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1) (cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1) 0.1 d L (cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1) (cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1) a (cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1)(cid:0)(cid:1) 0.0 0.0 0.2 0.4 0.6 dy FIG. 4: Phase diagram representing the typical equilibrium configurations of a system of model dynamins in the vicinity of a curved clathrin scaffold. c and c are the curvatures associated with the dynamins and clathrins, dy cl respectively, in units of the inverse membrane thickness a. In region G, the dynamins form a “gas” non interacting with the clathrin scaffold. In region L, the dynamins form a system of linear oligomers non interacting with the clathrin scaffold. In region R, the dynamins form a ring around the clathrin scaffold, which is reminiscent of real endocytosis. Sec. IIA) and of thermal agitation, we perform a Monte Carlo simulation. The details of the simulations are given in Appendix B. To prevent unphysicaldivergences of the elastic interactionenergy, it is necessary to introduce a hard-core steric repulsion preventing two inclusions to approach closer than a distance d. Since the size ofthe inclusions imprints is of the orderofthe membrane thickness a, we have chosend=2a. Actually, atsuchmicroscopicseparations,other short-rangedinteractionsintervene,the details ofwhichare still unknown. Here, we disregard them, since our interest lies in the mechanism by which the recruitment process and the formation of dynamin collars is driven. In a later stage, which we do not model here, dynamin rings are further stabilized by bio-chemical interactions [11]. Theresultsofthe MonteCarlosimulationsaresummarizedinthephasediagramofFig.4,intermsofthe curvaturesc andc ofthe clathrinsanddynaminsimprints,respectively. Here,wehavechosentospanc cl dy cl between0 to 0.2a−1: for a lattice constantb=3a andassuminga≃40˚A, this correspondsfor the clathrin- coatedbudtoamaximumcurvatureofradiusρ≃b/(ac )≃60nm. Sinceourclathrinpatchhas7spherical cl sources on its diameter, the size of the bud is 7b ≃ 85nm. These values are typical for clathrin-mediated endocytosis [6]. For the dynamins, we have spanned c between 0.1a−1 and 0.4a−1, which corresponds dy to a maximum curvature of the imprint ≃ 0.1nm−1. As for the membrane bending rigidity, we have taken κ=60k T, since for biological membranes at room temperature κ lies between 50 and 100 k T [40, 41]. B B The phase diagram displays three regimes (see Fig. 4): a state in which the dynamins are disordered in a gas-like fashion (G), a state in which the dynamins form linear oligomers that do not interact with the clathrinbud(L),andastateinwhichthedynaminsformaringaroundthe clathrinbud(R). Inregion(R), due to the shallowness of the dynamin imprints, the system is disordered by thermal agitation. Increasing the curvature of the dynamin imprints increases the elastic attraction between the dynamins (see Fig 3b) and leads to the formation of linear oligomers (L). These oligomerswrap aroundthe clathrin bud when the latter is sufficiently developed (R). Typical snapshots corresponding to the four points a, b, c, d in Fig. 4 are shown in Fig. 5. Note that in Fig. 5b the dynamin collar is rather “gaseous”due to the weakness of the dynamins’ imprints, while in Fig. 5d the ring is tight and well-ordered. It is difficult within the present paper to discuss the nature of the boundaries between the different “phases” displayed in Fig. 4. Because we are considering a rather small (thermodynamically speaking) number of dynamins, and because they are under the influence of a localized curvature field, the transition lines between the various regions of Fig. 4 are actually broad. Attempting to describe them as first- or second-order transition lines should not be meaningfull. 7 (a) (b) (c) (d) FIG. 5: Typical snapshots showing the equilibrium arrangement of model dynamins (bars) in the vicinity of the clathrin scaffold (hexagonal array). The figures (a), (b), (c) and (d) refer to the corresponding points in the phase diagram of Fig. 4. FIG.6: Membraneshapecorrespondingtopoint(d)inFigs.4and5,showingtheself-assemblyofaringofdynamins around a clathrin bud. IV. CONCLUSION In this paper, we haveshown that if membrane inserted dynamins produce cylindricalimprints and if the latter are sufficiently curved, then the resulting long-range forces mediated by the membrane elasticity are strongenoughtoovercomeBrownianmotionandbringthemintoacollararoundthe neckofaclathrinbud. Of course, simple diffusion could also bring dynamins around clathrin buds, and their binding into a ring could be the result of specific bio-chemical interactions. However, if a cylindrical imprint can speed up this process, then evolution may have selected it. Totestthismodel,onemightlookexperimentallyforlinearoligomersofdynamins(seeFig.5c). However, since “gaseous” rings are also possible (see Fig. 5b), the existence of such linear aggregates may not be necessary. It wouldbe more interesting to directly check,e.g., by cryo-electronmicroscopy,the shape of the dynamin region that penetrates within the membrane. Finally, note that our model is obviously over-simplified: i) many other integral proteins float around dynamins,ii)dynaminsmayinteractwithvariouslipidic domainswithinthe bilayer,iii)the membranemay have a spontaneous curvature due to its asymmetry, iv) fluctuations are not only thermal but also active, and hence could be larger than we estimate, and v) large values of the membrane tension could shorten the 8 range at which the dynamins are recruited (see Sec. I). Nonetheless, we believe that our model correctly captures the effects of the anisotropic elastic interactions. Acknowledgments We acknowledgefruitful discussions with R.Bruinsma,F. Ju¨licher,F. Képès,J.-M.Delosme, B.Goud, P. Chavrier, V. Norris, and J.-M. Valleton. APPENDIX A: MANY-BODY INTERACTIONS BETWEEN POINT-LIKE CURVATURE SOURCES LetusoutlinethederivationoftheinteractionbetweenN anisotropicpoint-likesourcesthatlocallyimprint acurvatureonthe membrane. As explainedinthetext, suchconstraintsmodelize awideclassofmembrane inclusions, including transmembrane and cytosol proteins partly inserted within the membrane. Forsmalldeformationsu(x,y)withrespecttothe(x,y)plane,thefreeenergyassociatedwiththecurvature elasticity of a membrane is given by [42]: F = κ dxdy ∇2u 2. (A1) el 2 Z Indeed, for small deformations, the Laplacian ∇2u(r) is(cid:0) equ(cid:1)al to the sum of the membrane’s principal curvatures at point r=(x,y). The material parameter κ is the bending rigidity. Determining the shape of the membrane in the presence of inclusions at positions r imprinting local α curvatures requires minimizing the elastic energy (A1) with local constraints on the membrane curvature tensor. In the small deformation limit, the elements of the latter are givenby the second spatial derivatives of the membrane shape: u (r), u (r) and u (r). Introducing 3N Lagrange multipliers Λα to enforce ,xx ,xy ,yy ij the curvature constraints, the Euler-Lagrangeequation corresponding to the constrained minimization is N ∇2∇2u(r)= Λα δ (r−r ) xx ,xx α αX=1h +Λα δ (r−r )+Λα δ (r−r ) , (A2) xy ,xy α yy ,yy α i where δ(r) is the two-dimensionalDirac’s delta anda comma indicates derivation. By linearity,the solution of this equation is 3N u(r)= Λ Γ (r), (A3) µ µ µ=1 X where the Λ ’s and Γ ’s are the 3N components of the column matrices µ µ Λ1 G (r−r ) xx ,xx 1 Λ1 G (r−r ) xy ,xy 1 Λ=Λ1yy, Γ(r)=G,yy(r−r1), (A4) Λ...2xx G,xx(r...−r2)     and 1 G(r)= r2lnr2 (A5) 16π is the Green function of the operator ∇2∇2, satisfying the equation ∇2∇2G(r)=δ(r). We introduce a column matrix K containing the values of the 3N constraints u (r ) ,xx 1 u (r ) ,xy 1 K=u,yy(r1). (A6) u (r )  ,xx 2   .   ..      9 With u(r) given by Eq. (A3), enforcing the constraints yields the following equation for the Lagrange multipliers: 3N M Λ =K , (A7) µν ν µ ν=1 X where the 3N ×3N matrix M is given by m m ... m 11 12 1N . m m .. M= 21 22 , (A8) ... ... ...   m ... ... m   N1 NN   in which the m ’s are N2 matrices of size 3×3 defined by αβ G (r ) G (r ) G (r ) ,xxxx βα ,xxxy βα ,xxyy βα m = G (r ) G (r ) G (r ) , (A9) αβ ,xxxy βα ,xxyy βα ,xyyy βα G (r ) G (r ) G (r )! ,xxyy βα ,xyyy βα ,yyyy βα where r =r −r . Setting βα α β r −r =r [cosθ xˆ+sinθ yˆ], (A10) α β βα αβ αβ yields explicitely cos(4θ )−2cos(2θ ) sin(2θ )[2cos(2θ )−1] −cos(4θ ) 1 αβ αβ αβ αβ αβ m = sin(2θ )[2cos(2θ )−1] −cos(4θ ) −sin(4θ )−sin(2θ ) . αβ 4πr2 αβ αβ αβ αβ αβ βα −cos(4θαβ) −sin(4θαβ)−sin(2θαβ) cos(4θαβ)+2cos(2θαβ)! (A11) Integrating Eq. (A1) by parts and taking into account the constraints yields the elastic energy 1 F = κKtM−1K, (A12) el 2 where Kt is the transpose of K. From Eqs. (A3) and (A7), the equilibrium shape of the membrane is given by u(r)=KtM−1Γ(r). (A13) When α = β, m as given by Eq. (A11) diverges: indeed Eq. (A1) correctly describes the membrane αβ elastic energy only for distances r >∼ r0, where r0 is of the order of the membrane thickness. It is therefore necessaryto introducea highwavevectorcutoffr−1 inthe theory. Fromthe definition ofthe Greenfunction 0 G(r), we deduce, in Fourier space d2q q4eiq·r G (r)= x . (A14) ,xxxx (2π)2 q4 Z Hence, introducing the cutoff, we obtain r0−1 qdq 2π 3 G (0)= dθ cos4θ = , (A15) ,xxxx (2π)2 32πr2 Z0 Z0 0 and similarly for the other elements of the matrix (A9). With the above prescription, we obtain 3 0 1 1 m = 0 1 0 . (A16) αα 32πr2 0 1 0 3! 10 As an illustration, let us consider the case of two identical isotropic inclusions, each prescribing the curvature c. Then Kt =(c,0,c,c,0,c), (A17) and, from Eqs. (A12), (A8), (A11), and (A16), with r =R, the interaction energy is 12 512πκ(r c)2 0 F = , (A18) el 4 2 R R +8 −32 r r (cid:18) 0(cid:19) (cid:18) 0(cid:19) in which we have discarded a constant term. Setting r = a/2, we indeed obtain the leading asymptotic 0 interaction (1). This special choice for r allows to match the result of Goulian et al. (1993), which was 0 obtained from multipolar expansions. It should be noted that the interaction given by Eq. (A18) is exact withinthepresentformalism(forrlargerthan≃a),whereasmultipolarexpansionscanonlygiveinanalytical form the leading asymptotic orders. When many inclusions are present, the matrixM and its inverse,which determines the interactionenergy through Eq. (A12), can be easily calculated numerically once the positions of the inclusions are defined. APPENDIX B: MONTE CARLO SIMULATIONS The Monte Carlo simulation that we perform employs the standard Metropolis algorithm [43]. 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